Immunomodulating heterocyclic compounds

ABSTRACT

Compounds of formula (I) are inhibitors of CD80 and useful in immunomodulation therapy: wherein R 1  and R 3  independently represent H; F; CI; Br; —NO 2 ; —CN; C 1 -C 6  alkyl optionally substituted by F or Cl; or C 1 -C 6  alkoxy optionally substituted by F; R 4  represents a carboxylic acid group (—COOH) or an ester thereof, or —C(═O)NR 6 R 7 , —NR 7 C(═O)R 6 , —NR 7 C(═O)OR 6 , —NHC(═O)NR 7 R 6  or —NHC(═S)NR 7 R 6  wherein R 6  represents H, or a radical of formula -(Alk) m -Q wherein m is 0 or 1, Alk is an optionally substituted divalent straight or branched C 1 -C 12  alkylene, or C 2 -C 12  alkenylene, or C 2 -C 12  alkynylene radical or a divalent C 3 -C 12  carbocyclic radical, any of which radicals may contain one or more —O—, —S— or —N(R 8 )— links wherein R 8 , represents H or C 1 -C 4  alkyl, C 3 -C 4  alkenyl, C 3 -C 4  alkynyl, or C 3 -C 6  cycloalkyl, and Q represents H; —NR 9 R 10  wherein R 9  and R 10  independently represents H; C 1 -C 4  alkyl; C 3 -C 4  alkenyl; C 3 -C 4  alkynyl; C 3 -C 6  cycloalkyl; an ester group; an optionally substituted carbocyclic or heterocyclic group; or R 9  and R 10  form a ring when taken together with the nitrogen to which they are attached, which ring is optionally substituted; and R 7  represents H or C 1 -C 6  alkyl; or when taken together with the atom or atoms to which they are attached R 6  and R 7  form an optionally substituted monocyclic heterocyclic ring having 5, 6 or 7 ring atoms; and X represents a bond or a divalent radical of formula -(Z) n -(Alk)- or -(Alk)-(Z) n -wherein Z represents —O—, —S— or —NH—, Alk is as defined in relation to R 6 , and n is 0 or 1

This application is a U.S. National Stage application of co-pending PCTapplication PCT/GB2004/001008 filed Mar. 10, 2004, which was publishedin English under PCT Article 21(2) on Sep. 23, 2004, and which claimsthe priority of Great Britain Patent Application No. 0305876.5, filedMar. 14, 2003 and Great Britain Patent Application No. 0319429.7, filedAug. 19, 2003, These applications are incorporated herein by referencein their entireties.

The present invention relates to novel heterocyclic compounds, tomethods for their preparation, to compositions containing them, and tomethods and use for clinical treatment of medical conditions which maybenefit from immunomodulation, e.g. autoimmune disease, rheumatoidarthritis, multiple sclerosis, diabetes, asthma, transplantation,systemic lupus erythematosis and psoriasis. More particularly thepresent invention relates to novel heterocyclic compounds, which areCD80 antagonists capable of inhibiting the interactions between CD80 andCD28, useful for immuno-inhibition.

BACKGROUND TO THE INVENTION

The immune system possesses the ability to control the homeostasisbetween the activation and inactivation of lymphocytes through variousregulatory mechanisms during and after an immune response. Among theseare mechanisms that specifically inhibit and/or turn off an immuneresponse. Thus, when an antigen is presented by MHC molecules to theT-cell receptor, the T-cells become properly activated only in thepresence of additional co-stimulatory signals. In the absence of theseaccessory signals there is no lymphocyte activation and either a stateof functional inactivation termed anergy or tolerance is induced, or theT-cell is specifically deleted by apoptosis.

One such co-stimulatory signal involves interaction of CD80 onspecialised antigen-presenting cells with CD28 on T-cells, and thissignal has been demonstrated to be essential for full T-cell activation.(Lenschow et al. (1996) Annu. Rev. Immunol., 14, 233-258). It wouldtherefore be desirable to provide compounds which inhibit this CD80/CD28interaction.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention there is provided a compound offormula (I) or a pharmaceutically or veterinarily acceptable salt,hydrate or solvate thereof:

wherein

-   R₁ and R₃ independently represent H; F; Cl; Br; —NO₂; —CN; C₁-C₆    alkyl optionally substituted by F or Cl; or C₁-C₆ alkoxy optionally    substituted by F;-   R₄ represents a carboxylic acid group (—COOH) or an ester thereof,    or —C(═O)NR₆R₇, —NR₇C(═O)R₆, —NR₇C(═O)OR₆, —NHC(═O)NR₇R₆ or    —NHC(═S)NR₇R₆ wherein    -   R₆ represents H, or a radical of formula -(Alk)_(m)-Q wherein        -   m is 0 or 1        -   Alk is an optionally substituted divalent straight or            branched C₁-C₁₂ alkylene, or C₂-C₁₂ alkenylene, or C₂-C₁₂            alkynylene radical or a divalent C₃-C₁₂ carbocyclic radical,            any of which radicals may contain one or more —O—, —S— or            —N(R₈)— links wherein R₈ represents H or C₁-C₄ alkyl, C₃-C₄            alkenyl, C₃-C₄ alkynyl, or C₃-C₆ cycloalkyl, and        -   Q represents H; —NR₉R₁₀ wherein R₉ and R₁₀ independently            represents H; C₁-C₄ alkyl; C₃-C₄ alkenyl; C₃-C₄ alkynyl;            C₃-C₆ cycloalkyl; an ester group; an optionally substituted            carbocyclic or heterocyclic group; or R₉ and R₁₀ form a ring            when taken together with the nitrogen to which they are            attached, which ring is optionally substituted; and    -   R₇ represents H or C₁-C₆ alkyl; or when taken together with the        atom or atoms to which they are attached R₆ and R₇ form an        optionally substituted monocyclic heterocyclic ring having 5, 6        or 7 ring atoms; and-   X represents a bond or a divalent radical of formula -(Z)_(n)-(Alk)-    or -(Alk)-(Z)_(n)-wherein Z represents —O—, —S— or —NH—, Alk is as    defined in relation to R₆ and n is 0 or 1.

Compounds (I) may exist in the form of tautomers, such as (I¹) and (I²):

Hereafter, the compounds of the invention may be represented andreferred to in any tautomeric form (I), and it is to be understood thatany and all tautomeric forms of structure (I), in particular (I¹) and(I²), are included in the invention.

Compounds of general formula (I) are CD80 antagonists. They inhibit theinteraction between CD80 and CD28 and thus the activation of T cells,thereby modulating the immune response.

Accordingly the invention also includes:

-   (i) a compound of formula (I) or a pharmaceutically or veterinarily    acceptable salt thereof for use in the treatment of conditions which    benefit from immunomodulation, and in particular for    immuno-inhibition.-   (ii) the use of a compound of formula (I) or a pharmaceutically or    veterinarily acceptable salt thereof in the manufacture of a    medicament for the treatment of conditions which benefit from    immunomodulation, and in particular for immuno-inhibition.-   (iii) a method of immunomodulation, and in particular    immuno-inhibition, in mammals, including humans, comprising    administration to a mammal in need of such treatment an    immunomodulatory effective dose of a compound of formula (I) or a    pharmaceutically or veterinarily acceptable salt thereof.-   (iv) a pharmaceutical or veterinary composition comprising a    compound of formula (I) or a pharmaceutically or veterinarily    acceptable salt thereof together with a pharmaceutically or    veterinarily acceptable excipient or carrier.    Conditions which Benefit from Immunomodulation Include:-   Acute disseminated encephalomyelitis-   Adrenal insufficiency-   Allergic angiitis and granulomatosis-   Amylodosis-   Ankylosing spondylitis-   Asthma-   Autoimmune Addison's disease-   Autoimmune alopecia-   Autoimmune chronic active hepatitis-   Autoimmune haemolytic anaemia-   Autoimmune Neutrogena-   Autoimmune thrombocytopenic purpura-   Behcet's disease-   Cerebellar degeneration-   Chronic active hepatitis-   Chronic inflammatory demyelinating polyradiculoneuropathy-   Chronic neuropathy with monoclonal gammopathy-   Classic polyarteritis nodosa-   Congenital adrenal hyperplasia-   Cryopathies-   Dermatitis herpetiformis-   Diabetes-   Eaton-Lambert myasthenic syndrome-   Encephalomyelitis-   Epidermolysis bullosa acquisita-   Erythema nodosa-   Gluten-sensitive enteropathy-   Goodpasture's syndrome-   Guillain-Barre syndrome-   Hashimoto's thyroiditis-   Hyperthyroidism-   Idiopathic hemachromatosis-   Idiopathic membranous glomerulonephritis-   Isolated vasculitis of the central nervous system-   Kawasaki's disease-   Minimal change renal disease-   Miscellaneous vasculitides-   Mixed connective tissue disease-   Multifocal motor neuropathy with conduction block-   Multiple sclerosis-   Myasthenia gravis-   Opsoclonus-myoclonus syndrome-   Pemphigoid-   Pemphigus-   pernicious anaemia-   Polymyositis/dermatomyositis-   Post-infective arthritides-   Primary biliary sclerosis-   Psoriasis-   Reactive arthritides-   Reiter's disease-   Retinopathy-   Rheumatoid arthritis-   Sclerosing cholangitis-   Sjogren's syndrome-   Stiff-man syndrome-   Subacute thyroiditis-   Systemic lupus erythematosis-   Systemic necrotizing vasculitides-   Systemic sclerosis (scleroderma)-   Takayasu's arteritis-   Temporal arteritis-   Thromboangiitis obliterans-   Type I and type II autoimmune polyglandular syndrome-   Ulcerative colitis-   Uveitis-   Wegener's granulomatosis

As used herein, the term “ester” refers to a group of the form —COOR,wherein R is a radical notionally derived from the alcohol ROH. Examplesof ester groups include the physiologically hydrolysable esters such asthe methyl, ethyl, n- and iso-propyl, n-, sec- and tert-butyl, andbenzyl esters.

As used herein the term “alkylene” refers to a straight or branchedalkyl chain having two unsatisfied valencies, for example —CH₂—,—CH₂CH₂—, —CH₂CH₂CH₂—, —CH(CH₃)CH₂—, —CH(CH₂CH₃)CH₂CH₂CH₂—, and—C(CH₃)₃.

As used herein the term “alkenylene” refers to a straight or branchedalkenyl chain having two unsatisfied valencies, for example —CH═CH—,—CH₂CH═CH—, —C(CH₃)═CH—, and —CH(CH₂CH₃)CH═CHCH₂—.

As used herein the term “alkynylene” refers to a straight or branchedalkynyl chain having two unsatisfied valencies, for example —C≡C—,—CH₂C≡C—, and —CH(CH₂CH₃)C≡CCH₂—.

Unless otherwise specified in the context in which it occurs, the term“substituted” as applied to any moiety herein means substituted with atleast one substituent, selected from, for example, (C₁-C₆)alkyl,(C₁-C₆)alkenyl, (C₂-C₆)alkynyl, fluoro-substituted(C₁-C₆)alkyl,fluoro-substituted(C₁-C₆)alkenyl, fluoro-substituted(C₂-C₆)alkynyl,(C₁-C₆)alkoxy and fluoro-substituted(C₁-C₆)alkoxy (including the specialcase where a ring is substituted on adjacent ring C atoms byalkylenedioxy such as methylenedioxy or ethylenedioxy),(C₁-C₆)alkylthio, phenyl, benzyl, phenoxy, benzyloxy, hydroxy, mercapto,amino, fluoro, chloro, bromo, cyano, nitro, oxo, —COOH, —SO₂OH, —CONH₂,—SO₂NH₂, —COR^(A), —COOR^(A), —SO₂OR^(A), —NHCOR^(A), —NHSO₂R^(A),—CONHR^(A), —SO₂NHR^(A), —NHR^(A), —NR^(A)R^(B), —CONR^(A)R^(B) or—SO₂NR^(A)R^(B) wherein R^(A) and R^(B) are independently a (C₁-C₆)alkylor (C₂-C₆)alkoxy group or a monocyclic carbocyclic or heterocyclic groupof from 5-7 ring members, or R^(A) and R^(B) form a ring when takentogether with the nitrogen to which they are attached. In the case where“substituted” means substituted by phenyl, benzyl, phenoxy, orbenzyloxy, the phenyl ring thereof may itself be substituted with any ofthe foregoing, except phenyl, benzyl, phenoxy, or benzyloxy.

As used herein the term “aryl” refers to a mono-, bi- or tri-cycliccarbocyclic aromatic radical, and to two such radicals covalently linkedto each other, Illustrative of such radicals are phenyl, biphenyl andnapthyl.

As used herein the unqualified term “carbocyclyl” or “carbocyclic”includes aryl, cycloalkyl and cycloalkenyl and refers to a ring system(monocyclic, bicyclic, tricyclic or bridged) whose ring atoms are allcarbon.

As used herein the unqualified term “cycloalkyl” refers to a carbocyclicring system which contains only single bonds between ring carbons.

As used herein the unqualified term “cycloalkenyl” refers to acarbocyclic ring system which contains at least one double bond betweena pair of ring carbons.

As used herein the term “heteroaryl” refers to a mono-, bi- ortri-cyclic aromatic radical containing one or more heteroatoms selectedfrom S, N and O. Illustrative of such radicals are thienyl, benzthienyl,furyl, benzfuryl, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl,benzthiazolyl, isothiazolyl, benzisothiazolyl, pyrazolyl, oxazolyl,benzoxazolyl, isoxazolyl, benzisoxazolyl, isothiazolyl, triazolyl,benztriazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl,pyrimidinyl, pyrazinyl, triazinyl, indolyl and indazolyl.

As used herein the unqualified term “heterocyclyl” or “heterocyclic”includes “heteroaryl” as defined above, and in particular means a mono-,bi- or tri-cyclic or bridged non-aromatic radical containing one or moreheteroatoms selected from S, N and O, and to groups consisting of amonocyclic non-aromatic radical containing one or more such heteroatomswhich is covalently linked to another such radical or to a monocycliccarbocyclic radical. Illustrative of such radicals are pyrrolyl,furanyl, tetrahydrofuranyl, thienyl, piperidinyl, imidazolyl, oxazolyl,isoxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl,pyrimidinyl, morpholinyl, piperazinyl, indolyl, morpholinyl,benzfuranyl, pyranyl, tetrahydropyranyl, quinuclidinyl, isoxazolyl,benzimidazolyl, methylenedioxyphenyl, ethylenedioxyphenyl, maleimido andsuccinimido groups.

Some compounds of the invention contain one or more chiral centresbecause of the presence of asymmetric carbon atoms. The presence ofasymmetric carbon atoms gives rise to stereoisomers or diastereoisomerswith R or S stereochemistry at each chiral centre. The inventionincludes all such stereoisomers and diastereoisomers and mixturesthereof.

Salts of salt forming compounds of the invention include physiologicallyacceptable acid addition salts and base salts Suitable acid additionsalts are formed from acids which form non-toxic salts. Examples includethe acetate, aspartate, benzoate, besylate, bicarbonate/carbonate,bisulphate/sulphate, borate, camsylate, citrate, edisylate, esylate,formate, fumarate, gluceptate, gluconate, glucuronate,hexafluorophosphate, hibenzate, hydrochloride/chloride,hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate,maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate,nicotinate, nitrate, orotate, oxalate, palmitate, pamoate,phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate, stearate,succinate, tartrate, tosylate and trifluoroacetate salts. Suitable basesalts are formed from bases which form non-toxic salts. Examples includethe aluminium, arginine, benzathine, calcium, choline, diethylamine,diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium,sodium, tromethamine and zinc salts.

Methods

Compounds of the invention wherein R₄ represents an amide group—C(═O)NR₆R₇ may be prepared by reaction of the appropriate amine HNR₆R₇with a compound of formula (II) to amidate the carboxylic acid group:

the symbols R₁, R₃, X, R₆ and R₇ being as defined in relation to formula(I) above.

Compounds (II) (ie compounds (I) of the invention wherein R₄ is acarboxylic acid group) may be prepared by reaction of a compound offormula (III) with a hydrazine of formula (IV):

This reaction may result in the preparation of a mixture of the positionisomers (IIA) and (IIB):

from which the desired isomer (IIA) may be separated.

Compounds (I) wherein R₄ is an ester or amide group may also be preparedfrom intermediate (III) by reaction with the appropriate hydrazine (IVA)

wherein R₄ is an ester or amide group. Again the reaction may result ina mixture of the ester or amide analogues of the carboxylic acids (IIA)and (IIB), from which the desired ester or amide isomer (I) may beseparated. Alternatively, the carboxylic acid compound (II) may simplybe esterified, or amidated.

Compounds (I) wherein R₄ is a “reverse amide” group —NR₇C(═O)R₆ may beprepared by Curtius rearrangement (see Ninomiya, K.; Shioiri, T.;Yamada, S. Tetrahedron (1974), 30(14), 2151-7) of the carboxylic acid(II) to the isocyanate (V)

followed by hydrolysis of the isocyanate group to an amino group andacylation of the amino group with, for example, the acid chlorideCl—C(═O)R₆. In cases where R₇ is not hydrogen, the R₇ substituent may beintroduced after the isocyanate reduction step or after the acylationstep.

In an alternative route to the “reverse amide” (R₄=—NR₇C(═O)R₆)compounds of the invention, a compound of structure (V) in which theisocyanate moiety is replaced by a nitro group may be reduced to thecorresponding amine, which may then be acylated to form the desiredreverse amide.

Compounds (I) wherein R₄ is a urea group —NHC(═O)NHR₆ or thiourea group—NHC(═S)NHR₆ may also be prepared from the isocyanate (V) or thecorresponding isothiocyanate by reaction with the appropriate amineH₂NR₆.

Compounds (I) wherein R₄ is a carbamate group —NR₇C(═O)OR₆ may beprepared by the reaction of the isocyanate with an appropriate alcoholR₆OH.

Further details of the synthetic methods for the preparation ofcompounds (I) of the invention, and intermediates such as (III), may befound in the examples herein.

In the Compounds of the Invention:

The radical R₄X— is preferably in the 4-position of the phenyl ring.

-   X may be, for example a bond, or a —CH₂— or —CH₂CH₂— radical. A bond    is presently preferred.-   R₃ may be, for example, H, F, Cl, methyl, methoxy, or    methylenedioxy. Currently it is preferred that R₃ is H.-   R₁ may be, for example, H, F, Cl, methyl, methoxy, or    methylenedioxy. Currently it is preferred that R₁ be hydrogen or    fluoro, particularly in the 6-position of the    3-oxo-1,3-dihydro-2H-pyrazolo[4,3-c]cinnolin-2-yl ring system.-   R₄ represents a carboxylic acid group (—COOH) or an ester thereof,    or —C(═O)NR₆R₇, —NR₇C(═O)R₆, —NR₇C(═O)OR₆ or —NHC(═O)NHR₆, all as    defined above.    -   When R₄ is an ester group, examples include those of formula        —COOR wherein R is methyl, ethyl n- or iso-propyl, n-, sec- or        tert-butyl, or benzyl ester.    -   R₆, when present, represents H, or a radical of formula        -(Alk)_(m)-Q wherein m, Alk and Q being as defined above. When m        is 1, Alk may be, for example a straight or branched C₁-C₆        alkylene radical, such as —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, and        —CH₂CH(CH₃)CH₂—. Alk may also be, for example, a divalent        cyclopropylene, cyclopentylene or cyclohexylene radical. The        radical Alk may be optionally substituted by, for example, OH,        oxo, CF₃, methoxy or ethoxy. The radical Alk may optionally        contain a hetero atom, for example in the form of an ether,        thioether or amino linkage.    -   The group Q may represent, for example, hydrogen; —NR₉R₁₀        wherein R₉ and R₁₀ may be the same or different and selected        from hydrogen, methyl, ethyl, n- or isopropyl or tert-butyl; an        ester group for example a methyl, ethyl or benzyl ester; or an        optionally substituted aryl, aryloxy, cycloalkyl, cycloalkenyl        or heterocyclic group, for example phenyl, phenoxy, cyclopentyl,        cyclohexyl, furyl, thienyl, quinuclidinyl, piperidyl, or        piperazinyl group.    -   R₇ when present represents H or C₁-C₆ alkyl, for example methyl,        ethyl n- or iso-propyl, n-, sec- or tert-butyl; or when taken        together with the atom or atoms to which they are attached R₆        and R₇ form a monocyclic heterocyclic ring having 5, 6 or 7 ring        atoms.

Especially preferred are the cases where R₄ represents —C(═O)NR₆R₇ or—NHC(═O)NR₇R₆ wherein R₇ is hydrogen and R₆ represents a radical offormula -(Alk)_(m)-Q wherein m is 1 and the divalent radical Alkcontains 3 or 4 carbon atoms and is unsubstituted, and Q represents—NR₉R₁₀ wherein R₉ and R₁₀ independently represents H; C₁-C₄ alkyl;C₃-C₄ alkenyl; C₃-C₄ alkynyl; C₃-C₆ cycloalkyl; an ester group; anoptionally substituted carbocyclic or heterocyclic group; or form a ringwhen taken together with the nitrogen to which they are attached, whichring is optionally substituted.

A specific preferred subset of compounds of the invention has formula(IC):

wherein X and R₄ are as specified above. In this subset, the radicalR₄X— may be in the 4-position of the phenyl ring. This subset includesin particular, compounds wherein X is a bond and R₄ is —C(═O)NR₆R₇wherein R₆ and R₇ are as specified above. For example, in such compoundsR₆ may be quinuclidinyl and R₇ hydrogen.

Specific compounds of the invention include those of the Examplesherein.

A preferred compound of the invention is4-(6-fluoro-3-oxo-1,3-dihydro-pyrazolo[4,3-c]cinnolin-2-yl)-N-(2,2-difluoro-ethyl)-benzamide,of formula (A)

or a pharmaceutically or veterinarily acceptable salt, hydrate orsolvate thereof.

Another preferred compound of the invention isN-[3-(tert-butyl-methyl-amino)-butyl]-4-(6-fluoro-3-oxo-1,3-dihydro-pyrazolo[4,3-c]cinnolin-2-yl)-benzamide,of formula (B):

or a pharmaceutically or veterinarily acceptable salt, hydrate orsolvate thereof.

As mentioned above, the invention includes pharmaceutical or veterinarycomposition comprising a compound of formula (I) or a pharmaceuticallyor veterinarily acceptable salt thereof together with a pharmaceuticallyor veterinarily acceptable excipient or carrier. In such compositions,it will be understood that the specific dose level for any particularpatient will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, route of administration, rate ofexcretion, drug combination and the cause and severity of the particulardisease undergoing therapy. Optimum dose levels and frequency of dosingwill be determined by clinical trial.

The compounds with which the invention is concerned may be prepared foradministration by any route consistent with their pharmacokineticproperties. The orally administrable compositions may be in the form oftablets, capsules, powders, granules, lozenges, liquid or gelpreparations, such as oral, topical, or sterile parenteral solutions orsuspensions. Tablets and capsules for oral administration may be in unitdose presentation form, and may contain conventional excipients such asbinding agents, for example syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinyl-pyrrolidone; fillers for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricant, for example magnesium stearate, talc, polyethylene glycol orsilica; disintegrants for example potato starch, or acceptable wettingagents such as sodium lauryl sulphate. The tablets may be coatedaccording to methods well known in normal pharmaceutical practice. Oralliquid preparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, for example sorbitol,syrup, methyl cellulose, glucose syrup, gelatin hydrogenated ediblefats; emulsifying agents, for example lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, fractionated coconut oil, oily esters such asglycerine, propylene glycol, or ethyl alcohol; preservatives, forexample methyl or propyl p-hydroxybenzoate or sorbic acid, and ifdesired conventional flavouring or colouring agents.

For topical application to the skin, the drug may be made up into acream, lotion or ointment. Cream or ointment formulations which may beused for the drug are conventional formulations well known in the art,for example as described in standard textbooks of pharmaceutics such asthe British Pharmacopoeia.

For topical application to the eye, the drug may be made up into asolution or suspension in a suitable sterile aqueous or non aqueousvehicle. Additives, for instance buffers such as sodium metabisulphiteor disodium edeate; preservatives including bactericidal and fungicidalagents such as phenyl mercuric acetate or nitrate, benzallkoniumchloride or chlorhexidine, and thickening agents such as hypromellosemay also be included.

The active ingredient may also be administered parenterally in a sterilemedium. Depending on the vehicle and concentration used, the drug caneither be suspended or dissolved in the vehicle. Advantageously,adjuvants such as a local anaesthetic, preservative and buffering agentscan be dissolved in the vehicle.

Embodiments of the invention are described in the following non-limitingExamples:

The following abbreviations are used in the experimental descriptions:

DMF Dimethyl formamide DMA Dimethyl acetamide DMSO Dimethyl sulphoxideHBTU O-Benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluoro-phosphate HPLC High performance liquid chromatography LCMS Liquidchromatography mass spectrum NMR Nuclear magnetic resonance spectroscopy

EXAMPLE 1 Step 1: Preparation of (henylhydrazono)malonic acid

Sodium mesoxalate monohydrate (5.00 g, 27.8 mmol) was dissolved in 1 Mhydrochloric acid (50 ml) to give a colourless cloudy solution.Phenylhydrazine (3.00 g, 2.72 ml, 27.8 mmol) was added dropwise at roomtemperature to the stirred mixture. A yellow precipitate formed, wascollected by filtration after 90 min and washed with water (50 ml). Thefilter cake was triturated with ethyl acetate/hexane [1:1], filtered anddried under vacuum. The title compound was isolated as a yellow powder(4.74 g, 22.7 mmol, 82%). LCMS: m/z 207 [M−H]⁺.

Alternatively the product can be extracted from the aqueous phase withethyl acetate (2×250 ml), the organic phase dried over magnesiumsulphate, filtered and the solvent removed under vacuum.

Step 2: Preparation of (phenylhydrazono)malonoyl dichloride

(Phenylhydrazono)malonic acid (1.00 g, 4.80 mmol) was mixed under inertatmosphere with dry chloroform (15 ml) to give a yellow suspension. Themixture was stirred at room temperature and phosphorus pentachloride(2.19 g, 10.5 mmol) was added portionwise. The reaction mixture washeated to reflux for 1.5 h to give a green solution. The mixture wascooled to room temperature and diluted with hexane (15 ml). A greenprecipitate formed, was collected by filtration and dried under vacuum.The title compound was isolated as a green powder (645 mg, 2.63 mmol,53%).

Step 3: Preparation of methyl 4-hydroxycinnoline-3-carboxylate

(Phenylhydrazono)malonoyl dichloride (2.45 g, 0.01 mmol) was mixed underinert atmosphere with 1,2-dichloroethane (15 ml) to give a yellowsuspension. Titanium tetrachloride (1.89 g, 1.09 ml) was added dropwiseto form a brown solution. The mixture was heated to reflux overnight,cooled to room temperature and quenched dropwise with methanol (15 ml).Stirring was continued for 30 min and volatiles were removed undervacuum. Water (100 ml) was added and the obtained suspension wasextracted with n-butanol (2×50 ml). The combined organic phases werewashed with water (2×20 ml) and concentrated under vacuum. The titlecompound was isolated as a green solid (1.04 g, 5.10 mmol, 51%). LCMS:m/z 205 [M+H]⁺.

Step 4: Preparation of methyl 4-chlorocinnoline-3-carboxylate

Thionyl chloride (8.15 g, 5 ml) was added dropwise under inertatmosphere to methyl 4-hydroxycinnoline-3-carboxylate (0.50 g, 2.45mmol). The mixture was heated to reflux for 1.5 h, cooled to roomtemperature and excess thionyl chloride was removed under vacuum.Toluene (5 ml) was added to the residue. The mixture was stirred at roomtemperature overnight. The solids were collected by filtration and driedunder vacuum. The title compound was isolated as a brown solid (248 mg,1.11 mmol, 45%). LCMS: m/z 223 [M+H]⁺.

Step 5: Preparation of4-(3-oxo-1,3-dihydro-2H-pyrazolo[4,3-c]cinnolin-2-yl)benzoic acid

4-Hydrazinobenzoic acid (68.4 mg, 0.45 mmol) was mixed at roomtemperature with ethanol (5 ml) to give a creme-coloured suspension.Methyl 4-chlorocinnoline-3-carboxylate (100 mg, 0.45 mmol) was added andthe mixture was heated to 45-50° C. for 1 h. The reaction mixture wascooled to room temperature and the solvent was removed under vacuum.Ethyl acetate (10 ml) was added to the residue. The mixture was stirredat room temperature for 1 h. The solids were collected by filtration anddried under vacuum. The title compound was isolated as a brown powder(120 mg, 0.39 mmol, 86%). LCMS: m/z 307 [M+H]⁺. NMR [DMSO-d₆]:δ=7.69-7.77 (m, 1H_(aryl)); 7.81-7.90 (m, 2H_(aryl)); 8.05 (d, J=8.85,2H_(aryl)); 8.20 (d, J=7.92 Hz, 1H_(aryl)); 8.33 (d, J=8.85 Hz,2H_(aryl)); 14.64 (s, NH).

Alternatively the reaction may be carried out at room temperature. Inthis case, a longer reaction time of 2-3 h may be required.

EXAMPLE 2 Preparation ofN-[(dimethylamino)propyl]-4-(3-oxo-1,3-dihydro-2H-pyrazolo[4,3-c]cinnolin-2-yl)benzamide

4-(3-oxo-1,3-dihydro-2H-pyrazolo[4,3-c]cinnolin-2-yl)benzoic acid (25mg, 0.08 mmol) was mixed with DMF (1 ml). Diisopropylethylamine (21 mg,28 μl, 0.16 mmol) and 3-dimethylaminopropylamine (8.2 mg, 10.0 μl, 0.09mmol) were added followed by HBTU (30.3 mg, 0.08 mmol). The mixture wasstirred at room temperature for 2 h. The product was purified bypreparative HPLC. The title compound was isolated as a red solid (12.6mg, 0.032 mmol, 40%). LCMS: m/z 391 [M+H]⁺.

EXAMPLE 3 Preparation ofN-benzyl-4-(3-oxo-1,3-dihydro-2H-pyrazolo[4,3-c]cinnolin-2-yl)benzamide

4-(3-oxo-1,3-dihydro-2H-pyrazolo[4,3-c]cinnolin-2-yl)benzoic acid (52mg, 0.17 mmol) was mixed with DMF (2 ml). Diisopropylethylamine (22 mg,29 μl, 0.17 mmol) and benzylamine (18.2 mg, 18.6 μl, 0.17 mmol) wereadded followed by HBTU (64.5 mg, 0.17 mmol). The mixture was stirred atroom temperature for 4 h. The product was purified by preparative HPLC.The title compound was isolated as a red solid (6.6 mg, 0.02 mmol, 10%).LCMS: m/z 396 [M+H]⁺.

EXAMPLE 4 Step 1: Preparation of4-(3-oxo-1,3-dihydro-2H-pyrazolo[4,3-c]cinnolin-2-yl)benzoyl chloride

Thionyl chloride (90 ml) was added to4-(3-oxo-1,3-dihydro-2H-pyrazolo-[4,3-c]cinnolin-2-yl)benzoic acid (2.36g, 7.70 mmol). The mixture was heated to reflux for 2 h under nitrogenatmosphere. A dark red solution was obtained, cooled to room temperatureand excess thionyl chloride was removed under vacuum. Toluene (30 ml)was added to the residues and the mixture was stirred at roomtemperature under nitrogen atmosphere until precipitation was complete.The solids were collected by filtration and washed with toluene (2×30ml). The title compound was isolated as a red solid (2.20 g, 6.77 mmol,88%) LCMS: m/z 321 [M+H]⁺ (methyl ester resulting from sample make-up inmethanol).

Step 2: Preparation ofN-[(cyclohexylamino)propyl]-4-(3-oxo-1,3-dihydro-2H-pyrazolo[4,3-c]cinnolin-2-yl)benzamide

4-(3-oxo-1,3-dihydro-2H-pyrazolo[4,3-c]cinnolin-2-yl)benzoyl chloride(97 mg, 0.30 mmol) was dissolved in anhydrous DMA (2 ml).Diisopropylethylamine (39 mg, 53 μl, 0.60 mmol) was added followed byN-cyclohexyl-1,3-propanediamine (52 mg, 0.60 mmol). The mixture wasstirred for 30 min. Water (5 ml) was added to give a dark redsuspension. The mixture was extracted with n-butanol (2×20 ml). Thecombined organic phases were washed with water and concentrated undervacuum until precipitation was observed. Hexane (20 ml) and ethylacetate (10 ml) were added, the solids were collected by filtration anddried under vacuum. The product was isolated as a dark red powder (82mg, 0.18 mmol, 62%). LCMS: m/z 445 [M+H]⁺.

EXAMPLE 5 Step 1: Preparation of [(2-Fluorophenyl)hydrazono]malonic acid

Sodium mesoxalate monohydrate (2.21 g, 12.3 mmol) was dissolved in 1 Mhydrochloric acid (50 ml) to give a colourless cloudy solution.2-Fluoro-phenylhydrazine hydrochloride (2.00 g, 12.3 mmol) was addedportionwise at room temperature to the stirred mixture. A yellowprecipitate formed, the mixture was diluted with water (50 ml) andstirring continued overnight. Ethyl acetate (150 ml) was added, thephases were mixed vigorously until the solids had dissolved. The phaseswere separated and the aqueous phase was washed with ethyl acetate (50ml). The combined organic phases were dried over magnesium sulfate,filtered and the solvent removed under vacuum. The title compound wasisolated as a yellow powder (2.55 g, 11.7 mmol, 92%). LCMS: m/z 227[M−H]⁺.

Step 2: Preparation of [(2-Fluorophenyl)hydrazono]malonoyl dichloride

(2-Fluorophenylhydrazono)malonic acid (1.33 g, 5.88 mmol) was mixedunder inert atmosphere with dry chloroform (20 ml) to give a yellowsuspension. The mixture was stirred at room temperature and phosphoruspentachloride (2.69 g, 12.9 mmol) was added portionwise. The reactionmixture was heated to reflux for 2 h to give a dark yellow solution. Themixture was cooled to room temperature and concentrated under vacuumuntil precipitation occurred. The solids were collected by filtration,washed with hexane (30 ml) and dried under vacuum. The title compoundwas isolated as a yellow powder (760 mg, 2.89 mmol, 49%).

Step 3: Preparation of methyl 8-fluoro-4-hydroxycinnoline-3-carboxylate

(2-Fluorophenylhydrazono)malonoyl dichloride (19.4 g, 74 mmol) was mixedunder inert atmosphere with 1,2-dichloroethane (100 ml) to give a yellowsuspension. Titanium tetrachloride (13.9 g, 8.08 ml, 74 mmol) was addeddropwise to form a brown solution. The mixture was heated to refluxovernight. Further titanium tetrachloride (13.9 g, 8.08 ml, 74 mmol) wasadded and heating continued for 24 h. The reaction mixture was cooled to0-5° C. and quenched dropwise with methanol (50 ml). Stirring wascontinued for 1 h at room temperature and volatiles were removed undervacuum. Water (300 ml) was added and the obtained suspension wasextracted with ethyl acetate (3×100 ml). The combined organic phaseswere dried over magnesium sulphate, filtered and concentrated undervacuum. A yellow solid was obtained (12 g crude product). LCMS: m/z 223[M+H]⁺.

Step 4: Preparation of4-(6-fluoro-3-oxo-1,3-dihydro-2H-pyrazolo[4,3-c]cinnolin-2-yl)benzoicacid

Crude 8-Fluoro-4-hydroxycinnoline-3-carboxylate from the previous stage(1.00 g, 4.95 mmol) was dissolved in thionyl chloride (50 ml). Thesolution was heated to reflux for 2-3 h until no further gas evolutionwas observed. The reaction mixture was cooled to room temperature andexcess thionyl chloride was removed under vacuum. The crude intermediatewas azeotroped with toluene (3×25 ml). A dark brown solid was obtained,which was taken up in ethanol (25 ml). 4-Hydrazinobenzoic acid (640 mg,4.21 mmol) was added and the mixture was stirred at room temperatureovernight. The solids were collected by filtration, slurried in 1 M HCl(100 ml), filtered, washed with hexane (50 ml) and dried under vacuum. Abrown solid was obtained (890 mg of crude product). LCMS: m/z [M+H]⁺325.

EXAMPLE 6 Step 1: Preparation of4-(6-fluoro-3-oxo-1,3-dihydro-2H-pyrazolo[4,3-c]cinnolin-2-yl)benzoicacid chloride

Crude4-(6-fluoro-3-oxo-1,3-dihydro-2H-pyrazolo[4,3-c]cinnolin-2-yl)-benzoicacid (1.45 g) from the previous stage was dissolved in thionyl chloride(50 ml). The mixture was heated to 70° C. for 2-3 h until no further gasevolution was observed. The mixture was cooled to room temperature andexcess thionyl chloride was removed under vacuum. The residues wereazeotroped with toluene (2×20 ml) to give a solid. The solid wascollected by filtration, washed with toluene and dried under vacuum. Theproduct was isolated as a yellow powder (670 mg, 1.95 mmol). LCMS: m/z[M+H]⁺ 339 (methyl ester resulting from sample make-up in methanol).

Step 2: Preparation of4-(6-fluoro-3-oxo-1,3-dihydro-2H-pyrazolo[4,3-c]cinnolin-2-yl)-N-(pyrrolidin-1-yl-butyl)benzamide

4-(6-fluoro-3-oxo-1,3-dihydro-2H-pyrazolo[4,3-c]cinnolin-2-yl)benzoylchloride (100 mg, 0.29 mmol) was dissolved in anhydrous DMA (2 ml).Diisopropylethylamine (75 mg, 101 μl, 0.58 mmol) was added followed by1-(4-aminobutyl)pyrrolidine (41 mg). The mixture was stirred at roomtemperature overnight. Water (5 ml) and n-butanol (5 ml) were added. Thephases were separated. The organic phase was washed with water (2×5 ml).The volatiles were removed under vacuum. The product was isolated as abrown powder (50 mg, 0.11 mmol, 37%). LCMS: m/z [M+H]⁺ 463.

EXAMPLE 7 Preparation of4-(6-fluoro-3-oxo-1,3-dihydro-2H-pyrazolo[4,3-c]cinnolin-2-yl)-N-(1,2,2,6,6-pentamethylpiperidine-4-yl)benzamide

4-(6-fluoro-3-oxo-1,3-dihydro-2H-pyrazolo[4,3-c]cinnolin-2-yl)benzoylchloride (100 mg, 0.29 mmol) was dissolved in anhydrous DMA (2 ml).Diisopropylethylamine (75 mg, 101 μl, 0.58 mmol) was added followed by4-amino-1,2,2,6,6-pentamethylpiperidine (49 mg, 0.29 mmol). The mixturewas stirred overnight. Water (5 ml) and n-butanol (5 ml) were added. Thephases were separated. The organic phase was washed with water (2×5 ml)and the solution was concentrated under vacuum. The title compound wasisolated as a dark red solid (50 mg, 0.105 mmol, 36%). LCMS: m/z [M+H]⁺477.

EXAMPLE 8 Step 1: Preparation of2-(4-nitrophenyl)-1,2-dihydro-3H-pyrazolo[4,3-c]cinnolin-3-one

Thionyl chloride (326 g, 200 ml) was added dropwise under inertatmosphere to methyl 4-hydroxycinnoline-3-carboxylate (10.0 g, 49 mmol).The mixture was heated to reflux for 2.5 h, cooled to room temperatureand excess thionyl chloride was removed under vacuum. Toluene (100 ml)was added to the residue and removed under vacuum. This procedure wasrepeated with further toluene (100 ml). A brown semi-solid material wasobtained and taken up in ethanol (200 ml). 4-Nitrophenylhydrazine (5.99g, 39.2 mmol) was added portionwise. The mixture was stirred at roomtemperature overnight. The mixture was heated to 40-45° C. for 1 h andcooled to room temperature. The solids were collected by filtration,triturated with ethanol (100 ml) and dried under vacuum. The titlecompound was isolated as a brown solid (8.42 g, 27.4 mmol, 70%). LCMS:m/z 308 [M+H]⁺.

Step 2: Preparation of2-(4-aminophenyl)-1,2-dihydro-3H-pyrazolo[4,3-c]cinnolin-3-one

2-(4-nitrophenyl)-1,2-dihydro-3H-pyrazolo[4,3-c]cinnolin-3-one (11.4 g,37.2 mmol) was suspended in a mixture of ethanol (100 ml) and water (100ml). Iron powder (11.1 g, 200 mmol) and ammonium chloride (5.34 g, 100mmol) were added. The mixture was heated to 80° C. overnight, cooled toroom temperature and basified with potassium carbonate to pH 9-10. Thesolids were removed by filtration through a pad of Celite®. The filtratewas extracted with n-butanol (2×200 ml). The combined organic phaseswere concentrated under vacuum to give a dark red solid. The solid wastriturated with methanol (100 ml), filtered and dried under vacuum. Thetitle compound was isolated as a dark red powder (5.58 g, 20.1 mmol,57%). LCMS: m/z 278 [M+H]⁺.

Step 3: Preparation ofN-[3-(dimethylamino)propyl]-N′-[4-(3-oxo-1,3-dihydro-2H-pyrazolo[4,3-c]cinnolin-2-yl)phenyl]urea

2-(4-aminophenyl)-1,2-dihydro-3H-pyrazolo[4,3-c]cinnolin-3-one (44 mg,0.16 mmol) was suspended in toluene under nitrogen atmosphere (0.5 ml)at 0-5° C. DMA (0.5 ml) was added followed by N,N′-carbonyldiimidazole(26 mg, 0.16 mmol). The mixture was stirred for 1 h at 0-5° C. beforemixed with a solution of 3-dimethylaminopropylamine (18 mg, 0.18 mmol)in toluene (0.5 ml). Stirring was continued for 1 h and the product waspurified by preparative HPLC. The title compound was isolated as a darkred powder (2.6 mg, 6 μmol, 4%). LCMS: m/z 406 [M+H]⁺.

EXAMPLE 9 Preparation of4-(3-oxo-1,3-dihydro-2H-pyrazolo[4,3-c]cinnolin-2-yl)benzoic acid ethylester.

The title compound was prepared by the method of Example 1 step 5,substituting 4-hydrazinobenzoic acid ethyl ester for the parent acid.MS: MH+=335.2

Results

The use of BIAcore Biomolecular Interaction Analysis

Biotinylated human CD80 (hCD80-BT) is a recombinant soluble form of amembrane bound receptor molecule (CD80) which binds to CD28 to initiateT cell activation. The interaction between CD80 and CD28 has beenextensively investigated (Collins et al, 2002). Biotinlyated humanHLA-A2-tax is the recombinant soluble form of a membrane bound receptormolecule that has been used in this example as a control protein, and isnot expected to interact with the compounds.

The BIAcore S51™ system was used for screening the compounds of Examples1-4 above. A series S sensor chip CM5 was docked onto the BIAcore S51™.Streptavidin was coupled to the carboxymethyl surface using standardamine coupling. The chip surface was activated with 0.2M EDC/0.05M NHS,followed by binding of streptavidin (0.25 mg/ml in 10 mM sodium acetatepH 5.0) and saturation of unoccupied sites with 1 M ethylenediamine.

The BIAcore S51 sensor chip has two separate sensor spots forimmobilisation of proteins. hCD80-BT was immobilised on thestreptavidin-coated surface of one sensor spot until a response ofapproximately 3000 RU was observed. A protein to control fornon-specific binding of the compound was immobilised on a second sensorspot. The control protein used for these experiments was a biotinylated,soluble form of the human HLA protein.

Dilution series of compounds (1000 nM-0.05 nM) were prepared in runningbuffer (10 mM, pH 7.4, 150 mM NaCl, 0.005% P20; 5% DMSO).

BIAcore S51™ was run at a flow rate of 30 μl/min using running buffer.Compounds and DMSO standard solutions for correction of data for solventeffects were injected. Data were recorded automatically and wereanalysed using BIAcore S51 Evaluation software.

The interaction between CD80 and the endogenous protein ligand (CD28) ishighly specific, but relatively weak, with a K_(D) of 4750 nM, and anoff-rate of greater than 0.2 s⁻¹. The compounds of Examples 2,3,4,6,7have greater affinity and longer residence times on CD80 than CD28,having K_(D)S of less than 100 nM, and off-rates of 2×10⁻², indicatingthat the cinnolines will be able to compete effectively with theendogenous ligand. The cinnolines showed no detectable interaction withthe control protein.

REFERENCES

Collins AV et al. (2002) Immunity 17, 201-210 “The interactionproperties of costimulatory molecules revisited”

Inhibition of Production of Interleukin-2 (IL-2) by Human Jurkat TCells.

Method

Human Raji cells were dispensed at a concentration of 2×10⁵ cells perwell in RPMI-1640 medium supplemented with 10% fetal calf serum, 1%penicillin/streptomycin, 1% glutamine (RPMI medium) in a 96-well roundbottom microtitre plate. Compounds under investigation (dissolved in100% DMSO) were diluted to eight-fold the desired final concentration inRPMI medium and added to the required final concentration for a totalvolume of 200 μl per well. After 20 minutes incubation at 37° C., JurkatT cells were added at a concentration of 2×10⁵ cells per well.Monoclonal antibody to CD3 (UCHT1, R&D Systems) was added to thecultures at a final concentration of 1 μg per ml, and where indicated,monoclonal antibody to CD28 (CD28.2, BD-Pharmingen) was also added at aconcentration of 2.5 μg per ml. Cells were cultured at 37° C. for 5hours, after which the plates were centrifuged and the supernatantsharvested for IL-2 ELISA assay using the IL-2 Eli-pair kit (DIACLONEResearch, Besancon, France) according to the manufacturers instructions.

By way of example, the compound of Example 2 (AV1142005) gave 65%inhibition at 30 μM.

Homogenous Time Resolved Fluorescence Assay

The examples described above were tested in a cell free Homogenous TimeResolved Fluorescence (HTRF) assay to determine their activity asinhibitors of the CD80-CD28 interaction.

In the assay, europium and allophycocyanin (APC) are associated withCD28 and CD80 indirectly (through antibody linkers) to form a complex,which brings the europium and APC into close proximity to generate asignal. The complex comprises the following six proteins: fluorescentlabel 1, linker antibody 1, CD28 fusion protein, CD80 fusion protein,linker antibody 2, and fluorescent label 2. The table below describesthese reagents in greater detail.

Fluorescent label 1 Anti-Rabbit IgG labelled with Europium (1 μg/ml)Linker antibody 1 Rabbit IgG specific for mouse Fc fragment (3 μg/ml)CD28 fusion protein CD28 - mouse Fc fragment fusion protein (0.48 μg/ml)CD80 fusion protein CD80 mouse Fab fragment (C215) fusion protein (1.9μg/ml) Linker antibody 2 GαMκ-biotin: biotinylated goat IgG specific formouse kappa chain (2 μg/ml) Fluorescent label 2 SA-APC: streptavidinlabelled allophycocyanin (8 μg/ml)

On formation of the complex, europium and APC are brought into proximityand a signal is generated.

Non-specific interaction was measured by substituting a mouse Fabfragment (C215) for the CD80 mouse Fab fragment fusion protein (1.9μg/ml). The assay was carried out in black 384 well plates in a finalvolume of 30 μl. Assay buffer: 50 mM Tris-HCl, 150 mM NaCl pH7.8,containing 0.1% BSA (w/v) added just prior to use.

Compounds were added to the above reagents in a concentration seriesranging between 100 μM-1.7 nM. The reaction was incubated for 4 hours atroom temperature. Dual measurements were made using a Wallac Victor 1420Multilabel Counter. First measurement: excitation 340 nm, emission 665nm, delay 50 μs, window time 200 μs. second measurement: excitation 340nm, emission 615 nm, delay 50 μs, window time 200 μs. Counts wereautomatically corrected for fluorescence crossover, quenching andbackground. The EC50 activities of compounds tested are recorded as:EC50: *=>10 μM,***=1-10 μM,**=<1 μM.

The compounds of Examples 1-8 had the following activities in the HTRFassay described above:

-   -   EXAMPLE 1*    -   EXAMPLE 2***    -   EXAMPLE 3***    -   EXAMPLE 4***    -   EXAMPLE 5*    -   EXAMPLE 6***    -   EXAMPLE 7***    -   EXAMPLE 8***    -   EXAMPLE 9**        Additional Examples

Further examples of compounds of the invention were synthesised bymethods analogous to those of Examples 1-8 above. The structures of thesynthesised compounds are shown in the following Table, together withtheir activities in the HTRF assay described above.

TABLE

Exam- ple MS No. X W R R′ MH+ Activity 9a. H — CH₂CH₂OMe H 364.2 ** 10.H —

H 446.2 *** 11. H — CH₂CH₂NMe₂ H 377.1 *** 12. H —

H 419.1 *** 13. H —

H 433.1 *** 14. H —

H 442.0 * 15. H — Ph H 382.0 ** 16. H —

H 463.0 * 17. H —

H 448.8 ** 18. H —

H 403.1 *** 19. H —

H 410.0 * 20. H —

H 411.0 *** 21. H —

H 441.2 ** 22. H —

H 431.1 ** 23. H —

H 414.1 *** 24. H —

H 402.2 ** 25. H —

H 418.4 * 26. H —

H 418.2 *** 27. H —

H 418.2 * 28. H —

H 417.9 ** 29. H —

H 378.0 *** 30. H —

H 445.2 *** 31. H —

H 479.0 ** 32. H —

H 445.2 *** 33. H —

H 376.2 ** 34. H —

H 420.0 ** 35. H —

H 400.0 ** 36. H —

H 418.0 * 37. H —

H 508.1 *** 38. H —

H 444.2 * 39. H —

H 441.1 ** 40. H —

H 467.2 ** 41. H —

H 424.1 ** 42. H —

H 496.9 ** 43. H —

H 404.1 ** 44. H —

H 480.0 * 45. H —

H 421.8 ** 46. H — Et H 334.2 *** 47. H —

H 425.0 *** 48. H — CH₂CH₂NHMe H 363.0 *** 49. H — CH₂CH₂NHEt H 377.1*** 50. H —

H 394.2 ** 51. H — CH₂CH₂OH H 350.2 *** 52. H — CH₂CH₂CH₂NHMe H 377.2*** 53. H — CH₂CH₂CH₂OiPr H 406.2 *** 54. H — CH₂CH₂CH₂CH₂NH₂ H 377.2*** 55. H —

H 390.2 *** 56. H —

H 414.1 ** 57. H —

H 388.2 ** 58. H — CH₂CH₂CH₂N(nBu)₂ H 475.2 *** 59. H — cyclododecyl H472.2 * 60. H — CH₂CH₂NEt₂ H 405.1 *** 61. H —

H 417.2 *** 62. H —

H 402.2 ** 63. H — CH₂CH₂OPh H 426.0 ** 64. H —

H 480.2 ** 65. H —

H 475.2 ** 66. H —

H 406.1 ** 67. H — CH₂CH₂CH₂OnBu H 420.0 *** 68. H —

H 459.3 *** 69. H —

H 417.3 *** 70. H —

H 362.3 *** 71. H —

H 362.3 *** 72. H — CH(Et)₂ H 376.3 ** 73. H — CH₂CH₂CH₂CH₂Ph H 438.4 **74. H —

H 492.2 ** 75. H —

H 416.3 ** 76. H —

H 411.2 ** 77. H — CH₂CH₂SEt H 394.2 *** 78. H — Cyclopropyl H 346.2 **79. H —

H 417.3 *** 80. H —

H 479.3 *** 81. H —

H 447.2 *** 82. H — CH₂CH₂CH(CH₃)CH₃ H 376.2 ** 83. H — cyclopentyl H374.2 ** 84. H — nPropyl H 348.2 ** 85. H — CH₂CH₂tBu H 390.3 ** 86. H —

H 479.3 *** 87. H — CH₂cycloheptyl H 416.4 * 88. H —

H 390.3 ** 89. H —

H 376.3 *** 90. H —

H 480.2 ** 91. H —

H 477.1 *** 92. H —

H 432.4 * 93. H —

H 420.1 ** 94. H —

H 465.3 *** 95. H —

H 411.4 *** 96. H —

H 404.3 ** 97. H —

H 463.0 ** 98. H —

H 465.4 ** 99. H —

H 434.4 ** 100. H —

H 400.3 * 101. H —

H 518.4 *** 102. H —

H 418.4 ** 103. H —

H 445.4 *** 104. H —

H 461.4 ** 105. H —

H 438.4 * 106. H —

H 394.3 ** 107. H —

H 376.3 ** 108. H —

H 391.4 *** 109. H —

H 393.4 *** 110. H —

H 405.5 *** 111. H — CH₂CH₂CH₂OH H 364.4 ** 112. H — CH₂CH₂CH₂CH₂CH₂OH H392.4 *** 113. H — nHexyl H 390.4 ** 114. H —

H 489.4 ** 115. H —

H 378.4 ** 116. H —

H 406.4 * 117. H —

H 505.5 ** 118. H —

H 406.4 ** 119. H —

H 378.4 ** 120. H —

H 416.4 ** 121. H —

H 442.4 ** 122. H —

H 442.4 * 123. H NH

H 494.3 ** 124. H NH

H 405.3 * 125. H NH

H 432.3 ** 126. H NH

H 429.3 * 127. H NH

H 403.3 * 128. H NH CH₂CH₂CH₂OEt H 407.2 ** 129. H NH

H 461.3 *** 130. H NH CH₂CH₂NMe₂ H 392.2 *** 131. H NH allyl H 361.3 ***132. H NH

H 434.3 *** 133. H NH CH₂CH₂CH₂OMe H 393.2 ** 134. H NH

H 460.3 *** 135. H NH

H 474.3 ** 136. H NH

H 420.1 * 137. H NH

H 449.2 ** 138. H NH

H 377.3 ** 139. H NH iPr H 363.3 ** 140. H NH CH₂CH₂OMe H 379.3 ** 141.H NH CH₂CH₂NHiPr H 406.2 *** 142. H NH CH₂CH₂NHMe H 378.2 *** 143. H NHCH₂CH₂NHEt H 392.2 *** 144. H NH CH₂CH₂NHnPr H 406.2 *** 145. H NHCH₂CH₂OCH₂CH₂OH H 409.2 *** 146. H NH CH₂CH₂OH H 365.2 *** 147. H NHCH₂CH₂Ph H 425.3 ** 148. H NH CH₂CH₂CH₂NHiPr H 420.2 *** 149. H NHCH₂CH₂CH₂OiPr H 421.2 ** 150. H NH CH₂CH₂CH₂OH H 379.2 *** 151. H NHCH₂CH₂CH₂CH₂CH₂OH H 407.2 ** 152. H NH

H 490.1 * 153. H NH

H 405.3 ** 154. H NH

H 393.1 ** 155. H NH

H 470.3 ** 156. H NH

H 421.2 ** 157. H NH

H 378.1 ** 158. H NH

H 421.1 ** 159. H NH CH₂CH₂CH₂OC₁₂H₂₅ H 547.3 *** 160. H NHCH₂CH₂CH₂OnBu H 435.2 * 161. H NH CH₂CH₂CH₂SMe H 409.2 ** 162. H NH

H 432.3 *** 163. H NH

H 519.9 ** 164. H NH

H 461.2 * 165. H NH

H 375.2 ** 166. H NH

H 405.2 ** 167. H NH

H 377.3 ** 168. H NH

H 462.4 *** 169. H NH

H 430.3 *** 170. H NH CH₂CH₂CHO H 377.2 * 171. H NH

H 393.3 *** 172. H NH

H 494.3 ** 173. H NH

H 391.3 ** 174. H NH

H 393.2 ** 175. H NH

H 435.2 ** 176. H — CH₂CH₂CH₂NEt₂ H 419.4 *** 177. H NH nBu H 377.4 **178. H NH CH₂CH₂SMe H 395.3 ** 179. H NH

H 448.4 *** 180. H NH

H 523.3 * 181. H NH

H 419.4 * 182. H NH

H 464.3 ** 183. H NH

H 418.4 *** 184. H NH

H 426.3 ** 185. H NH

H 434.4 *** 186. H NH

H 460.4 *** 187. H NH CH(Et)₂ H 391.4 ** 188. H NH CH₂CH₂CH₂CH₂Ph H453.4 * 189. H NH

H 507.5 ** 190. H NH

H 419.4 ** 191. H NH

H 406.4 ?? 192. H NH

H 435.4 * 193. H NH

H 454.5 *** 194. H NH

H 431.5 * 195. H NH

H 405.4 ** 196. H NH

H 426.4 ** 197. H NH

H 494.5 ** 198. H NH

H 405.4 ** 199. H NH

H 415.5 * 200. H NH CH₂CH₂SCH₂Ph H 471.4 * 201. H NH

H 457.5 * 202. H NH

H 457.4 * 203. H NH

H 391.4 * 204. H NH CH₂cycloheptyl H 431.5 * 205. H NH

H 435.4 * 206. H NH CH₂CH₂N(nBu)₂ H 476.5 *** 207. H NH

H 405.4 ** 208. H NH CH₂CH₂OPh H 441.4 ** 209. H NH

H 433.4 * 210. H NH

H 393.4 *** 211. H NH

H 495.5 * 212. H NH

H 509.4 * 213. H NH

H 478.5 ** 214. H NH

H 480.4 *** 215. H NH

H 435.4 * 216. H NH

H 449.4 * 217. H NH

H 426.4 ** 218. H NH

H 419.5 * 219. H NH

H 540.5 ** 220. H NH

H 492.5 * 221. H NH

H 405.5 * 222. H NH

H 434.4 *** 223. H NH

H 449.4 ** 224. H NH

H 522.5 *** 225. H NH

H 471.4 * 226. H NH

H 437.4 * 227. H NH

H 576.4 * 228. H NH

H 446.4 *** 229. H NH

H 432.4 *** 230. H NH

H 383.3 * 231. H NH

H 429.4 *** 232. 6-F — CH₂CH₂CH₂NMe₂ H 409.4 *** 233. 6-F —

H 449.4 *** 234. 6-F —

H 477.4 *** 235. 6-F —

H 463.4 *** 236. H —

H 456.4 * 237. H —

H 439.4 *** 238. H —

H 390.3 ** 239. H — cycloButyl H 360.4 ** 240. H —

H 426.4 *** 241. H — nButyl H 362.4 ** 242. H —

H 386.4 *** 243. H — iPr H 348.4 *** 244. H —

H 402.4 ** 245. H — nHeptyl H 404.4 ** 246. H — Allyl H 346.3 *** 247. H— CH₂CH₂CH₂OMe H 378.4 *** 248. H —

H 464.3 * 249. H —

H 464.3 * 250. H —

H 414.3 *** 251. H — nPentyl H 376.4 * 252. H —

H 422.3 * 253. H —

H 442.3 * 254. H —

H 508.2 * 255. H —

H 416.3 * 256. H —

H 403.4 ** 257. H —

H 456.4 * 258. H —

H 362.3 ** 259. H —

H *** 260. H —

H ** 261. H NH

H 431.5 * 262. H NH

H 405.4 * 263. H NH

H 391.4 ** 264. H NH

H 518.5 * 265. H NH

H * 266. H —

H 511.4 *** 267. H —

H 391.4 ** 268. H —

H 449.4 ** 269. H — CH₂CH₂NHnPr H 391.4 *** 270. H — CH₂CH₂Ph H 410.4 *271. H — CH₂CH₂CH₂CH₂CH₂CH₂NH₂ H 405.4 *** 272. H —

H 414.4 * 273. H — CH₂CH₂CH₂OC₁₂H₂₅ H 532.6 * 274. H — CH₂CH₂CH₂SCH₃ H394.4 *** 275. H —

H 446.4 ** 276. H — CH(Et)CH₂OCH₂Ph H 468.4 ** 277. H —

H 390.3 ** 278. H —

H 415.4 *** 279. H — CH₂CH₂NHnBu H 405.4 *** 280. H — CH₂CH₂NHCH₂CH₂NEt₂H 448.5 *** 281. H — CH₂CH₂NHCH₂Ph H 439.4 *** 282. H NH Et H 349.4 ***283. H NH

H 457.4 * 284. H NH

H 429.3 * 285. H NH

H 415.3 *** 286. H NH

H 455.3 * 287. H NH

H 433.4 *** 288. H NH

H 522.5 *** 289. H NH

H 431.3 ** 290. H NH

H 418.4 *** 291. H NH CH₂CH₂CH₂Ph H 439.4 * 292. H NH

H 495.4 * 293. H NH

H 454.3 * 294. H NH

H 533.5 ** 295. H NH CH₂CH₂CH₂NHCH₃ H 392.4 *** 296. H NH

H 456.4 * 297. H NH

H 446.4 ** 298. H NH

H 429.4 *** 299. H NH

H 417.5 * 300. H NH

H 433.5 *** 301. H NH

H 460.5 *** 302. H NH

H 486.4 * 303. H NH

H 476.4 * 304. H NH CH(CH₃)CH₂CH₂Ph H 453.4 * 305. H NH

H 433.5 * 306. H NH CH₂CH(OMe)₂ H 409.4 *** 307. H NH CH₂CH(OEt)₂ H437.5 ** 308. H NH CH₂CH(CH₃)CH₂CH₃ H 391.4 ** 309. H NH CH(CH₃)CH₂CH₃ H377.4 ** 310. H NH

H 432.4 *** 311. H — CH₂CHF₂ H 370.4 *** 312. H — CH₂CH₂CF₃ H 402.4 ***313. H —

H 440.5 ** 314. H —

H 412.5 *** 315. H —

H 422.5 ** 316. H —

H 402.5 ** 317. H —

H 416.5 ** 318. H —

H 442.5 *** 319. H — tBu H 362.5 *** 320. H — CH₂Si(CH₃)₃ H 392.5 * 321.H — CH(CH₃)CH₂CH₂CH₃ H 376.5 *** 322. H — CH(CH₃)CH₂CH₂CH₂CH₃ H 390.5*** 323. 6-F —

H 497.6 *** 324. 6-F NH CH₂CH₂N(CH₃)₂ H 410.5 *** 325. 8-F —

H 449.3 *** 326. 8-F — CH₂CH₂N(Et)₂ H 423.3 *** 327. 8-F —

H 435.3 *** 328. 8-F —

H 497.3 *** 329. 8-F — CH₂CH₂CH₂N(Bu)₂ H 493.4 *** 330. 8-F —CH₂CH₂CH₂N(Et)₂ H 437.3 *** 331. 8-F —

H 435.5 *** 332. 8-F —

H 463.3 *** 333. 6-F — CH₂CH═CHCH₃ H 378.2 *** 334. 6-F —

H 525.3 *** 335. 6-F —

H 479.3 *** 336. 6-F —

H 517.4 *** 337. 6-F —

H 511.3 *** 338. H —

H 447.3 *** 339. H —

H 445.3 *** 340. 6-F —

H 378.2 *** 341. 6-F — CH₂CH₂NHnPr H 409.3 *** 342. 6-F — CH₂CH₂N(Et)₂ H423.3 *** 343. 6-F —

H 435.3 *** 344. 6-F — CH₂CH₂NHnBu H 423.3 *** 345. 6-F —CH₂CH₂CH₂N(nBu)₂ H 493.4 *** 346. 6-F — CH₂CH₂CH₂N(Et)₂ H 437.3 *** 347.6-F — CH₂CH₂NHCH₂Ph H 457.3 *** 348. 6-F — CH₂CH₂CH₂NHiPr H 423.3 ***349. 6-F —

H 421.3 *** 350. 6-F —

H 451.3 *** 351. 6-F — CH₂CH₂CH₂CH₂NH₂ H 395.3 *** 352. 6-F —

H 435.3 *** 353. 6-F —

H 433.3 *** 354. 6-F — CH₂CH₂CH₂OnBu H 438.3 *** 355. 6-F —CH₂CH₂CH₂NHMe H 395.3 *** 356. 6-F — CH₂CH₂NHMe H 381.3 *** 357. 6-F —CH₂CH₂NHEt H 395.3 *** 358. 6-F —

H 463.4 *** 359. 6-F —

H 481.3 *** 360. 6-F —

H 462.2 ** 361. 6-F —

H 488.3 *** 362. 6-F —

H 442.3 ** 363. 6-F —

H 462.2 ** 364. 6-F —

H 462.2 ** 365. 6-F —

H 506.2,508.2 * 366. 6-F —

H 442.3 ** 367. 6-F —

H 442.3 ** 368. 6-F —

H 506.2,508.2 ** 369. 6-F —

H 480.2 ** 370. 6-F —

H 496.2 ** 371. 6-F —

H 496.2 ** 372. 6-F —

H 496.2 ** 373. 6-F —

H 446.3 ** 374. 6-F —

H 463.3 *** 375. 6-F — tBu H 380.3 *** 376. 6-F — CH₂CHF₂ H 388.2 ***377. 6-F — CH₂CH═CH₂ H 364.2 *** 378. 6-F —

H 553.4 *** 379. 6-F —

H 524.4 ** 380. 6-F —

H 575.3 *** 381. 8-F — CH₂CH₂CH₂N(Me)₂ H 409.3 *** 382. 8-F —

H 463.3 *** 383. 8-F — CH₂CH₂NHEt H 409.3 *** 384. 8-F — CH₂CH₂NHBu H423.3 *** 385. 8-F — CH₂CH₂CH₂NHiPr H 423.3 *** 386. 8-F —CH₂CH₂CH₂CH₂OH H 396.3 *** 387. 9-F — CH₂CH₂CH₂N(Me)₂ H 409.2 *** 388.9-F —

H 449.2 *** 389. 9-F —

H 463.3 *** 390. 9-F —

H 477.3 *** 391. 9-F —

H 421.2 *** 392. 9-F — CH₂CH₂CH₂N(Et)₂ H 437.2 *** 393. 9-F —

H 435.2 *** 394. 9-F —

H 463.2 *** 395. 9-F — CH₂CH₂CH₂NHiPr H 423.2 *** 396. 9-F —CH₂CH₂CH₂NHMe H 395.2 *** 397. 9-F —

H 451.2 *** 398. 9-F — CH₂CH₂CH₂N(nBu)₂ H 493.3 *** 399. 9-F —

H 483.2 *** 400. 9-F — tBu H 380.2 *** 401. 9-F —

H 433.2 *** 402. 9-F —

475.2 ** 403. 9-F —

H 463.3 *** 404. 9-F —

H 437.2 *** 405. 9-F —

H 421.2 *** 406. 8- — CH₂CH₂CH₂N(Me)₂ H 405.3 *** Me 407. 8-Me —

H 459.3 *** 408. 8-Me —

H 473.4 *** 409. 8-Me —

H 445.3 *** 410. 6-F —

H 421.3 *** 411. 6-Cl — CH₂CH₂CH₂N(Me)₂ H 425.3 *** 412. 6-Cl —

H 479.2 *** 413. 6-Cl —

H 465.3 *** 414. 6,8- — CH₂CH₂CH₂N(Me)₂ H 427.3 *** diF 415. 6,8-diF —

H 481.3 *** 416. 6,8-diF —

H 467.2 *** 417. 6-F —

H *** 418. 8- — CH₂CH₂CH₂NHMe H 407.2 *** MeO 419. 6-F —

H 481.2 *** 420. 6-F —

H 437.2 *** 421. 6-F —

475.2 ** 422. 6-F — CH₂CF₂CF₂CF₃ H 456.1 *** 423. 6-F — CH₂CH₂CF₃ H420.1 *** 424. 6-F —

H 378.1 *** 425. 6-F —

H 392.2 *** 426. 6-F — CH₂CH₂F H 370.1 *** 427. 8-F —

H 477.3 *** 428. 6,9- — CH₂CH₂CH₂NHMe H 413.2 *** diF 429. 6,9-diF —

H 481.3 *** 430. 6,9-diF —

H 467.2 *** 431. 6,9-diF —

H 495.3 *** 432. 6-F — CH₂CH₂CH₂CH₂N(Et)Me H 437.2 *** 433. 6-F —CH₂CH₂CH₂CH₂N(Et)₂ H 451.3 *** 434. 6-F —

H 465.2 *** 435. 6-F — CH₂CH₂CH₂CH₂N(Me)CH₂CH═CH₂ H 449.2 *** 436. 6-F —

H 491.4 *** 437. 6-F — CH₂CH₂CH₂CH₂F H 398.2 *** 438. 6-F —

H 471.2 ** 439. 6-F —

H 463.3 *** 440. 6-F —

H 407.2 *** 441. 6-F —

H 409.3 *** 442. 6-F — CH₂CH₂CH₂NHnPr H 423.2 *** 443. 6-F —

H 409.2 *** 444. 6-F —

H 421.1 *** 445. 6-F — CH₂CH₂CH₂NH₂ H 381.2 *** 446. 8-Cl —CH₂CH₂CH₂N(Me)₂ H 425.2 *** 447. 8-Cl —

H 493.2 *** 448. 8-Cl —

H 479.3 *** 449. 8-Cl —

H 465.2 *** 450. 6-F — Et H 352.2 *** 451. 6-F —

H 463.3 *** 452. 6-F — Et nPr 394.2 ** 453. 6-F —

H 435.2 *** 454. 6-F —

H 477.3 *** 455. 6-F — CH₂tBu H 394.2 *** 456. 6-F —

H 503.3 *** 457. 6-F —

H 539.3 *** 458. 6-F —

H 471.2 *** 459. 6-F — CH₂CH₂CH₂CH₂N(CH₂CH═CH₂)₂ H 475.3 *** 460. 6-F —

H 493.3 *** 461. 6-F —

422.2 *** 462. 6-F —

H 477.3 *** 463. 6-F —

406.2 *** 464. 6-F —

H 477.3 *** 465. 6-F —

406.2 *** 466. 6-F —

H 519.2 *** 467. 6-F — CH₂CF₂CF₂H H 438.1 *** 468. 6-F —

H 491.3 *** 469. 6-F —

H 503.3 *** 470. 6-F —

H 531.3 *** 471. 6-F —

H 481.2 *** 472. 6-F —

H 449.3 *** 473. 8-F — CH₂CF₂H H 388.1 *** 474. 6-F — allyl allyl 404.2** 475. 6-F — CH₂CH₂CF═CF₂ H 432.1 ** 476. 6-F —

H 507.3 *** 477. 6-F —

H 491.3 *** 478. 6-F —

H 465.3 *** 479. 6-F —

H 451.2 *** 480. 6-F —

H 465.2 *** 481. 6-F —

H 465.2 *** 482. 6-F —

H 363.1 *** 483. 6-F —

H 461.3 *** 484. 6-F —

H 515.3 *** 485. 6-F —

H 416.2 *** 486. 6-F —

H 377.1 *** 487. 6-F — C(CH₂OH)₃ H 428.2 *** 488. 6-F —

H 393.1 *** 489. 6-F —

421.2 *** 490. 6-F —

407.1 *** 491. 6-F — CH₂CONH₂ H 381.2 *** 492. 6-F —

H 454.1 *** 493. 6-F —

H 398.1 *** 494. 6-F —

420.2 ** 495. 6-F —

446.2 496. 6-F —

446.2 497. 6-F —

H 439.1 ** 498. 6-F —

H 419.2 *** 499. 6-F —

H 421.2 *** 500. 6,9- — CH₂CHF₂ H 406.2 *** diF 501. 6-F —

H 422.2 *** 502. 6-F —

H 396.2 *** 503. 6-F —

H 450.2 ** 504. 6-F —

H 436.2 ** 505. 6-F — CH₂CH₂NHCH₂CH₂OH H 411.2 *** 506. 6-F —

H 464.2 ** 507. 6-F — CH(CH₂OH)₂ H 398.1 *** 508. 6-F — CH(CH₃)CH₂OH H382.1 *** 509. 6-F — CH(CH₂CH₃)CH₂OH H 396.2 *** 510. 6-F —

H 456.2 ** 511. 6-F —

H 474.1 ** 512. 6-F —

H 520.2 * 513. 6-F —

H 474.1 ** 514. 6-F —

H 458.2 *** 515. 6-F —

H 497.2 ** 516. 6-F — C(CH₃)₂CH₂OH H 396.2 * 517. 6-F — C CH₃(CH₂OH)₂ H412.1 ***

Examples of the result of testing the above compounds in the assay forinhibition of production of interleukin-2 (IL-2) by human Jurkat Tcells, described above, are as follows:

Compound Example No (see Concentration Percentage Inhibition table) (μM)(relative to DMSO = 0%) 478 10 56.0 376 10 56.7 353 10 77.4 429 10 58.8349 10 79.5 68 10 71.7 235 10 59.3 288 30 72 162 30 54.4 350 10 74.2 38110 48.5 442 10 58.9 482 10 39.2 472 10 58.4 453 10 55.7 53 30 63.8

1. A compound of formula (I) or a pharmaceutically or veterinarilyacceptable salt or hydrate thereof:

wherein R₁, and R₃ independently represent H; F; Cl; Br; —NO₂; —CN;C₁-C₆ alkyl optionally substituted by F or Cl; or C₁-C₆ alkoxyoptionally substituted by F; R₄ represents a carboxylic acid group(—COOH) or an ester thereof, or —C(═O)NR₆R₇, —NR₇C(═O)R₆, —NR₇C(═O)OR₆,—NHC(═O)NR₇R₆ or —NHC(═S)NR₇R₆ wherein R₆ represents H, or a radical offormula -(Alk)_(m)-Q wherein m is 0 or 1 Alk is an optionallysubstituted divalent straight or branched C₁-C₁₂ alkylene, or C₂-C₁₂alkenylene, or C₂-C₁₂ alkynylene radical or a divalent C₃-C₁₂carbocyclic radical, any of which radicals may contain one or more —O—,—S— or —N(R₈)— links wherein R₈ represents H or C₁-C₄ alkyl, C₃-C₄alkenyl, C₃-C₄ alkynyl, or C₃-C₆ cycloalkyl, and Q represents H; —NR₉R₁₀wherein R₉ and R₁₀ independently represents H; C₁-C₄ alkyl; C₃-C₄alkenyl; C₃-C₄ alkynyl; C₃-C₆ cycloalkyl; an ester group; an optionallysubstituted carbocyclic or heterocyclic group; or R₉ and R₁₀ form a ringwhen taken together with the nitrogen to which they are attached, whichring is optionally substituted; and R₇ represents H or C₁-C₆ alkyl; orwhen taken together with the atom or atoms to which they are attached R₆and R₇ form an optionally substituted monocyclic heterocyclic ringhaving 5, 6 or 7 ring atoms; and X represents a bond or a divalentradical of formula -(Z)_(n)-(Alk)- or -(Alk)-(Z)_(n)-wherein Zrepresents —O—, —S— or —NH—, Alk is as defined in relation to R₆ and nis 0 or
 1. 2. A compound as claimed in claim 1 wherein the radical R₄X—is in the 4-position of the phenyl ring.
 3. A compound as claimed inclaim 1 wherein X is a bond.
 4. A compound as claimed in claim 1 whereinR₃ is hydrogen.
 5. A compound as claimed in claim 1 wherein R₁ ishydrogen or fluoro.
 6. A compound as claimed in claim 1 wherein R₄represents —C(═O)NR₆R₇.
 7. A compound as claimed in claim 1 wherein R₄represents —NHC(═O)NR₇R₆.
 8. A compound as claimed in claim 7 wherein R₆is a quinuclidinyl radical.
 9. A compound as claimed in claim 1 whereinR₆ represents a radical of formula -(Alk)_(m)-Q wherein m is 1 and thedivalent radical Alk contains 3 or 4 carbon atoms and is unsubstituted,and Q represents —NR₉R₁₀ wherein R₉ and R₁₀ independently represent H;C₁-C₄ alkyl; C₃-C₄ alkenyl; C₃-C₄ alkynyl; C₃-C₆ cycloalkyl; an estergroup; an optionally substituted carbocyclic or heterocyclic group; orform a ring when taken together with the nitrogen to which they areattached, which ring is optionally substituted.
 10. A compound asclaimed in claim 6 wherein R₇ is hydrogen.
 11. A compound as claimed inclaim 1 wherein Q represents H; —CF₃; —OH; —SH; —NR₈R₈wherein each R₈independently represents H; C₁-C₄ alkyl; C₃-C₄ alkenyl; C₃-C₄ alkynyl;C₃-C₆ cycloalkyl; an ester group; an optionally substituted aryl,aryloxy, cycloalkyl, cycloalkenyl or heterocyclic group; or form a ringwhen taken together with the nitrogen to which they are attached; and R₇represents H or C₁-C₆ alkyl; or when taken together with the atom oratoms to which they are attached R₆ and R₇ form a monocyclicheterocyclic ring having 5, 6 or 7 ring atoms.
 12. A compound as claimedin claim 11 wherein R₄ represents a carboxylic acid group (—COOH) or anester group of formula —COOR wherein R is methyl, ethyl, n- oriso-propyl, n-, sec- or tert-butyl or benzyl.
 13. A compound as claimedin claim 11 wherein R₆ represents a radical of formula -(Alk)_(m)-Qwherein m is 1, Alk is —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, or —CH₂CH(CH₃)CH₂—,or a divalent cyclopropylene, cyclopentylene or cyclohexylene radical,optionally substituted by —OH, oxo, CF₃, methoxy or ethoxy, and Qrepresents hydrogen; —NR₈R₈ wherein each R₈ may be the same or differentand selected from hydrogen, methyl, ethyl, n- or isopropyl ortert-butyl; a methyl, ethyl or benzyl ester; or an optionallysubstituted phenyl, phenoxy, cyclopentyl, cyclohexyl, furyl, thienyl,piperidyl, or piperazinyl group.
 14. A compound as claimed in claim 11wherein R₇ represents methyl, ethyl, n- or iso-propyl, n-, sec- ortert-butyl; or when taken together with the atom or atoms to which theyare attached R₆ and R₇ form a monocyclic heterocyclic ring having 5, 6or 7 ring atoms.
 15. A compound as claimed in claim 11 wherein R₁ is H,F, Cl, methyl, methoxy, or methylenedioxy.
 16. A compound as claimed inclaim 11 wherein R₁ is F, in the 6-position of the3-oxo-1,3-dihydro-2H-pyrazolo[4,3-c]cinnolin-2-yl ring system.
 17. Acompound as claimed in claim 11 wherein R₃ is H, F, Cl, methyl, methoxy,or methylenedioxy.
 18. A compound as claimed in claim 11 wherein X is abond, or a —CH₂— or —CH₂CH₂— radical.
 19. A compound of formula (IC) ora pharmaceutically or veterinarily acceptable salt or hydrate thereof:

wherein X is a bond, or a —CH₂— or —CH₂CH₂— radical and R₄ is acarboxylic acid group (—COOH), an ester group of formula —COOR wherein Ris methyl, ethyl, n- or iso-propyl, n-, sec- or tert-butyl or benzyl, or—NHC(═O)NR₆R₇ wherein R₆ represents H, or a radical of formula-(Alk)_(m)-Q wherein m is 0 or 1 Alk is an optionally substituteddivalent straight or branched C₁-C₁₂ alkylene, or C₂-C₁₂ alkenylene, orC₂-C₁₂ alkynylene radical or a divalent C₃-C₁₂ carbocyclic radical, anyof which radicals may contain one or more —O—, —S— or —N(R₈)— linkswherein R₈ represents H or C₁-C₄ alkyl, C₃-C₄, alkenyl, C₃-C₄ alkynyl,or C₃-C₆ cycloalkyl, and Q represents H; —NR₉R₁₀ wherein R₉ and R₁₀independently represents H; C₁-C₄alkyl; C₃-C₄ alkenyl, C₃-C₄alkynyl;C₃-C₆ cycloalkyl; an ester group; an optionally substituted carbocyclicor heterocyclic group; or R₉ and R₁₀ form a ring when taken togetherwith the nitrogen to which they are attached, which ring is optionallysubstituted; and R₇ represents H or C₁-C₆alkyl; or when taken togetherwith the atom or atoms to which they are attached R₆ and R₇ form anoptionally substituted monocyclic heterocyclic ring having 5, 6 or 7ring atoms.
 20. A compound as claimed in claim 18 wherein the radicalR₄X— is in the 4-position of the phenyl ring.
 21. A compound as claimedin claim 19 wherein X is a bond and R₄ is —C(═O)NR₆R₇.
 22. The compound4-(6-fluoro-3-oxo-1,3-dihydro-pyrazolo[4,3-c]cinnolin-2-yl)-N-(2,2-difluoro-ethylyl)-benzamide,of formula (A)

or a pharmaceutically or veterinarily acceptable salt or hydratethereof.
 23. The compoundN-[3-(tert-butyl-methyl-amino)-butyl]-4-(6-fluoro-3-oxo-1,3-dihydro-pyrazolo[4,3-c]cinnolin-2-yl)-benzamide,of formula (B):

or a pharmaceutically or veterinarily acceptable salt or hydratethereof.
 24. A pharmaceutical or veterinary composition comprising acompound as claimed in claim 1 together with a pharmaceutically orveterinarily acceptable excipient or carrier.